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Background Heart Rate Variability (HRV) is a valid, scalable biomarker of stress. We aimed to examine associations between HRV and psychosocial outcomes in adolescents and young adults (AYAs) with cancer. Methods This was a secondary analysis of baseline data from a randomized trial testing a resilience intervention in AYAs with cancer. Two widely used HRV metrics, the standard deviation of normal to normal beats (SDNN) and root mean square of successive differences (RMSSD), were derived from electrocardiograms. Patient-reported outcome (PRO) survey measures included quality of life, anxiety, depression, distress, and resilience. Linear regression models were used to test associations between HRV and PRO scores. The Wilcoxon rank sum test was used to test differences in median HRV values among participant subgroups. Results Among the n = 76 patients with available electrocardiograms, the mean age was 16 years (SD 3y), 63% were white, and leukemia/lymphoma was the most common diagnosis. Compared to healthy adolescents, AYAs with cancer had lower median HRV (SDNN [Females: 31.9 (12.8–50.7) vs 66.4 (46.0–86.8), pConclusion In this secondary analysis, we did not find an association between HRV and psychosocial PROs among AYAs with cancer. HRV measures were lower than for healthy adolescents. Larger prospective studies in AYA biopsychosocial research are needed.

Although Plasmodium vivax parasites are the predominant cause of malaria outside of sub-Saharan Africa, they not always prioritised by elimination programmes. P. vivax is resilient and poses challenges through its ability to re-emerge from dormancy in the human liver. With observed growing drug-resistance and the increasing reports of life-threatening infections, new tools to inform elimination efforts are needed. In order to halt transmission, we need to better understand the dynamics of transmission, the movement of parasites, and the reservoirs of infection in order to design targeted interventions. The use of molecular genetics and epidemiology for tracking and studying malaria parasite populations has been applied successfully in P. falciparum species and here we sought to develop a molecular genetic tool for P. vivax. By assembling the largest set of P. vivax whole genome sequences (n = 433) spanning 17 countries, and applying a machine learning approach, we created a 71 SNP barcode with high predictive ability to identify geographic origin (91.4%). Further, due to the inclusion of markers for within population variability, the barcode may also distinguish local transmission networks. By using P. vivax data from a low-transmission setting in Malaysia, we demonstrate the potential ability to infer outbreak events. By characterising the barcoding SNP genotypes in P. vivax DNA sourced from UK travellers (n = 132) to ten malaria endemic countries predominantly not used in the barcode construction, we correctly predicted the geographic region of infection origin. Overall, the 71 SNP barcode outperforms previously published genotyping methods and when rolled-out within new portable platforms, is likely to be an invaluable tool for informing targeted interventions towards elimination of this resilient human malaria., Author summary Plasmodium vivax is the most widespread parasite causing human malaria, with more than one-third of the world’s population being at risk of infection. P. vivax is resilient due to its dormant liver phase, and there are increasing reports of drug-resistance and life-threatening infections. Despite this, P. vivax malaria is not always prioritised by elimination programmes. New molecular tools are needed to inform elimination efforts, including through better understanding the geographical source and outbreaks of P. vivax, thereby leading to the halting of transmission and the targeting of reservoirs of infection. Our work describes a 71 genetic marker barcode for P. vivax that has high predictive ability to identify the geographic origin, and has the potential to distinguish local transmission networks. If the 71 genetic marker barcode is implemented within new portable molecular platforms, it is likely to be an invaluable tool for informing targeted interventions towards elimination of this resilient human malaria.

The survival of an organism depends on the ability to make adaptive decisions to achieve the needs of the organism: where to get food, who to mate with, and how to evade predators. Decision-making is a term used to describe a collection of behavioral and/or computational functions that guide the selection of an option amongst a set of alternatives. Some of these functions may include calculating the costs and benefits of a particular action, evaluating differences in value of each of the alternative outcomes and the likelihood of receiving a particular outcome, using past experiences to generate predictions or expectations about action-outcome associations, and/or integration of past experiences to make novel inferences that can be used in new environments. There is considerable interest in understanding the neurobiological mechanisms that mediate these decision-making functions and recent advances in behavioral approaches, neuroscience techniques, and neuroimaging measures have begun to develop mechanistic links between biology, reward, and decision making. This multidisciplinary work holds great promise for elucidating the biological mechanisms mediating decision-making deficits in normal and abnormal states. The multidisciplinary studies included in this Collection provide new insights into the neuroscience of decision making and reward.

Background Colorectal cancer (CRC) is among the most common cancers in economically developed countries and developing world. While dietary factors are associated with risk of CRC in the West and urban China, little is known about risk or protective factors in rural China. Methods The Linxian General Population Nutrition Intervention Trial (NIT) cohort was established over 30 years ago to test whether daily multivitamin/mineral supplements could reduce the incidence and mortality of esophageal/gastric cardia cancer. The cohort included a total of 29,553 healthy participants 40–69 years old who were randomly assigned to supplements or placebos via a 24 fractional factorial study design. We examined risk factors for the development of CRC as well as the effects of four different nutritional factors (Factor A: retinol, zinc; B: riboflavin, niacin; C: ascorbic acid, molybdenum; D: selenium, alpha-tocopherol, beta-carotene,) on CRC incidence following 5.25 years of supplementation in this randomized, placebo-controlled intervention trial. Results CRC risk increased with age and height as well as piped water usage, family history of CRC, and consumption of foods cooked in oil, eggs, and fresh fruits. No effect on CRC was seen for any of these four intervention factors tested in both genders, but CRC was reduced 37% in females who received Factor D (selenium/alpha-tocopherol/beta-carotene) (RR = 0.63, 95% CI = 0.43–0.92, P = 0.016) compared to females who did not receive Factor D. Conclusions In this undernourished rural Chinese population, CRC risk factors in this Chinese cohort showed both similarities and differences compared to Western and urban Asian Chinese populations. Intervention results suggested a potential benefit for women supplemented with selenium/alpha-tocopherol/beta-carotene.

Background The leading cause of mortality for patients with the neurofibromatosis type 1 (NF1) cancer predisposition syndrome is the development of malignant peripheral nerve sheath tumor (MPNST), an aggressive soft tissue sarcoma. In the setting of NF1, this cancer type frequently arises from within its common and benign precursor, plexiform neurofibroma (PN). Transformation from PN to MPNST is challenging to diagnose due to difficulties in distinguishing cross-sectional imaging results and intralesional heterogeneity resulting in biopsy sampling errors. Methods and findings This multi-institutional study from the National Cancer Institute and Washington University in St. Louis used fragment size analysis and ultra-low-pass whole genome sequencing (ULP-WGS) of plasma cell-free DNA (cfDNA) to distinguish between MPNST and PN in patients with NF1. Following in silico enrichment for short cfDNA fragments and copy number analysis to estimate the fraction of plasma cfDNA originating from tumor (tumor fraction), we developed a noninvasive classifier that differentiates MPNST from PN with 86% pretreatment accuracy (91% specificity, 75% sensitivity) and 89% accuracy on serial analysis (91% specificity, 83% sensitivity). Healthy controls without NF1 (participants = 16, plasma samples = 16), PN (participants = 23, plasma samples = 23), and MPNST (participants = 14, plasma samples = 46) cohorts showed significant differences in tumor fraction in plasma (P = 0.001) as well as cfDNA fragment length (P < 0.001) with MPNST samples harboring shorter fragments and being enriched for tumor-derived cfDNA relative to PN and healthy controls. No other covariates were significant on multivariate logistic regression. Mutational analysis demonstrated focal NF1 copy number loss in PN and MPNST patient plasma but not in healthy controls. Greater genomic instability including alterations associated with malignant transformation (focal copy number gains in chromosome arms 1q, 7p, 8q, 9q, and 17q; focal copy number losses in SUZ12, SMARCA2, CDKN2A/B, and chromosome arms 6p and 9p) was more prominently observed in MPNST plasma. Furthermore, the sum of longest tumor diameters (SLD) visualized by cross-sectional imaging correlated significantly with paired tumor fractions in plasma from MPNST patients (r = 0.39, P = 0.024). On serial analysis, tumor fraction levels in plasma dynamically correlated with treatment response to therapy and minimal residual disease (MRD) detection before relapse. Study limitations include a modest MPNST sample size despite accrual from 2 major referral centers for this rare malignancy, and lack of uniform treatment and imaging protocols representing a real-world cohort. Conclusions Tumor fraction levels derived from cfDNA fragment size and copy number alteration analysis of plasma cfDNA using ULP-WGS significantly correlated with MPNST tumor burden, accurately distinguished MPNST from its benign PN precursor, and dynamically correlated with treatment response. In the future, our findings could form the basis for improved early cancer detection and monitoring in high-risk cancer-predisposed populations., Jeffrey J. Szymanski and colleagues investigate the use of cell-free DNA ultra-low-pass whole genome sequencing to distinguish the malignant peripheral nerve sheath tumor (MPNST) from its benign precursor lesion in patients with Neurofibromatosis type 1 in United States., Author summary Why was this study done? Neurofibromatosis type 1 (NF1) is the most common inherited cancer predisposition syndrome. The leading cause of mortality in NF1 is malignant peripheral nerve sheath tumor (MPNST), an aggressive soft tissue sarcoma that arises from a benign plexiform neurofibroma (PN) precursor lesion. Transformation from PN to MPNST is challenging to detect by imaging (due to difficulty in distinguishing PN from MPNST radiologically) or by biopsy (due to intralesional heterogeneity), which often delays the diagnosis of MPNST and results in a worsened prognosis. What did the researchers do and find? We conducted a multi-institutional study involving 2 large NF1 referral centers, the National Cancer Institute and Washington University in St. Louis, involving 53 patients from whom plasma cell-free DNA (cfDNA) was analyzed using ultra-low-pass whole genome sequencing (ULP-WGS). We found that cfDNA from patients with MPNST harbors a shorter fragmentation profile compared to patients with PN or healthy donors. Using sequencing reads from this fragmentation profile, we quantified genome-wide copy number alterations (CNAs) in cfDNA and used CNAs to estimate the fraction of plasma cfDNA originating from tumor. Tumor fraction in plasma cfDNA distinguished pretreatment MPSNT from PN with 86% accuracy. Plasma cfDNA from MPNST and PN patients harbored focal copy number loss of NF1 not found in healthy donors. Strikingly, MPNST patient cfDNA also had significantly greater tumor genomic instability compared to PN, with CNAs in key genomic loci previously observed in MPNST tissue (i.e., gain of chromosome arm 8q and loss of 9p), which enabled sensitive and specific liquid biopsy discrimination of MPNST from PN. Plasma-derived tumor fraction correlated with tumor size from imaging in MPNST patients, and serial cfDNA analysis demonstrated the potential for noninvasive detection of minimal residual disease, treatment response assessment, and the potential for even greater assay sensitivity. What do these findings mean? Our findings suggest that cfDNA fragment analysis followed by ULP-WGS has the potential to be developed as a biomarker for treatment response and as a screening assay for early detection of MPNST. This study provides, to our knowledge, the first evidence for the ability of liquid biopsy to distinguish between benign and malignant tumors in a heritable cancer predisposition syndrome.

Successful reproduction is critical to the persistence of at-risk species; however, reproductive characteristics are understudied in many wild species. New Zealand’s endemic tuatara (Sphenodon punctatus), the sole surviving member of the reptile order Rhynchocephalia, is restricted to 10% of its historic range. To complement ongoing conservation efforts, we collected and characterized mature sperm from male tuatara for the first time. Semen collected both during mating and from urine after courting contained motile sperm and had the potential for a very high percentage of viable sperm cells (98%). Scanning electron microscopy revealed a filiform sperm cell with distinct divisions: head, midpiece, tail, and reduced end piece. Finally, our initial curvilinear velocity estimates for tuatara sperm are 2–4 times faster than any previously studied reptile. Further work is needed to examine these trends at a larger scale; however, this research provides valuable information regarding reproduction in this basal reptile.

Characterising connectivity between geographically separated biological populations is a common goal in many fields. Recent approaches to understanding connectivity between malaria parasite populations, with implications for disease control efforts, have used estimates of relatedness based on identity-by-descent (IBD). However, uncertainty around estimated relatedness has not been accounted for. IBD-based relatedness estimates with uncertainty were computed for pairs of monoclonal Plasmodium falciparum samples collected from five cities on the Colombian-Pacific coast where long-term clonal propagation of P. falciparum is frequent. The cities include two official ports, Buenaventura and Tumaco, that are separated geographically but connected by frequent marine traffic. Fractions of highly-related sample pairs (whose classification using a threshold accounts for uncertainty) were greater within cities versus between. However, based on both highly-related fractions and on a threshold-free approach (Wasserstein distances between parasite populations) connectivity between Buenaventura and Tumaco was disproportionally high. Buenaventura-Tumaco connectivity was consistent with transmission events involving parasites from five clonal components (groups of statistically indistinguishable parasites identified under a graph theoretic framework). To conclude, P. falciparum population connectivity on the Colombian-Pacific coast abides by accessibility not isolation-by-distance, potentially implicating marine traffic in malaria transmission with opportunities for targeted intervention. Further investigations are required to test this hypothesis. For the first time in malaria epidemiology (and to our knowledge in ecological and epidemiological studies more generally), we account for uncertainty around estimated relatedness (an important consideration for studies that plan to use genotype versus whole genome sequence data to estimate IBD-based relatedness); we also use threshold-free methods to compare parasite populations and identify clonal components. Threshold-free methods are especially important in analyses of malaria parasites and other recombining organisms with mixed mating systems where thresholds do not have clear interpretation (e.g. due to clonal propagation) and thus undermine the cross-comparison of studies., Author summary In this study we aimed to characterise connectivity between populations of Plasmodium falciparum malaria parasites sampled from five cities on the Colombian-Pacific coast where long-term clonal propagation of P. falciparum is frequent. We found that connectivity along the coast is consistent with accessibility not isolation-by-distance, potentially implicating marine traffic in malaria transmission and thus presenting a possible opportunity for targeted intervention. Our study makes methodological contributions that could be adapted to analyses of other recombining organisms. Akin to numerous studies in both epidemiology and ecological, to characterise connectivity, we used genetic data and computed estimates of relatedness based on identity-by-descent (IBD). However, unlike previous studies, confidence intervals around relatedness estimates were included in our analyses. This is an important consideration for all studies that plan to use limited genetic data to estimate IBD-based relatedness. To identify groups of clonal parasites and to compare parasite populations across cities, we used methods that avoid thresholds, e.g. of highly-related parasite pairs. Threshold-free methods promote cross-comparison in studies of recombining organisms for which thresholds do not have a clear interpretation (e.g. for malaria parasites, where the frequency of clonal propagation varies in space and time and is not fully understood).

BackgroundHendra virus (HeV) infection is endemic in Australian flying-fox populations. Habitat loss has increased the peri-urban presence of flying-foxes, increasing the risk of contact and therefore viral 'spillovers' into horse and human populations. An equine vaccine is available and horse-husbandry practices that minimize HeV exposure are encouraged, but their adoption is suboptimal. Ecological approaches-such as habitat creation and conservation-could complement vaccination and behavioural strategies by reducing spillover risks, but these are controversial.MethodsWe convened three community juries (two regional; one metropolitan) to elicit the views of well-informed citizens on the acceptability of adding ecological approaches to current interventions for HeV risk. Thirty-one participants of diverse backgrounds, mixed genders and ages were recruited using random-digit-dialling. Each jury was presented with balanced factual evidence, given time to ask questions of expert presenters and, after deliberation, come to well-reasoned conclusions.ResultsAll juries voted unanimously that ecological strategies should be included in HeV risk management strategies but concluded that current interventions-including vaccination and changing horse-husbandry practices-must remain the priority. The key reasons given for adopting ecological approaches were: (i) they address underlying drivers of disease emergence, (ii) the potential to prevent spillover of other bat-borne pathogens, and (iii) there would be broader community benefits. Juries differed regarding the best mechanism to create/conserve flying-fox habitat: participants in regional centres favoured direct government action, whereas the metropolitan jury preferred to place the burden on landholders.ConclusionsInformed citizens acknowledge the value of addressing the drivers of bat-borne infectious risks but differ substantially as to the best implementation strategies. Ecological approaches to securing bat habitat could find broad social support in Australia, but disagreement about how best to achieve them indicates the need for negotiation with affected communities to co-develop fair, effective and locally appropriate policies.

Objective To present treatment coverage rates and risk factors associated with uncorrected refractive error in Australia. Methods Thirty population clusters were randomly selected from all geographic remoteness strata in Australia to provide samples of 1738 Indigenous Australians aged 40 years and older and 3098 non-Indigenous Australians aged 50 years and older. Presenting visual acuity was measured and those with vision loss (worse than 6/12) underwent pinhole testing and hand-held auto-refraction. Participants whose corrected visual acuity improved to be 6/12 or better were assigned as having uncorrected refractive error as the main cause of vision loss. The treatment coverage rates of refractive error were calculated (proportion of participants with refractive error that had distance correction and presenting visual acuity better than 6/12), and risk factor analysis for refractive correction was performed. Results The refractive error treatment coverage rate in Indigenous Australians of 82.2% (95% CI 78.6–85.3) was significantly lower than in non-Indigenous Australians (93.5%, 92.0–94.8) (Odds ratio [OR] 0.51, 0.35–0.75). In Indigenous participants, remoteness (OR 0.41, 0.19–0.89 and OR 0.55, 0.35–0.85 in Outer Regional and Very Remote areas, respectively), having never undergone an eye examination (OR 0.08, 0.02–0.43) and having consulted a health worker other than an optometrist or ophthalmologist (OR 0.30, 0.11–0.84) were risk factors for low coverage. On the other hand, speaking English was a protective factor (OR 2.72, 1.13–6.45) for treatment of refractive error. Compared to non-Indigenous Australians who had an eye examination within one year, participants who had not undergone an eye examination within the past five years (OR 0.08, 0.03–0.21) or had never been examined (OR 0.05, 0.10–0.23) had lower coverage. Conclusion Interventions that increase integrated optometry services in regional and remote Indigenous communities may improve the treatment coverage rate of refractive error. Increasing refractive error treatment coverage rates in both Indigenous and non-Indigenous Australians through at least five-yearly eye examinations and the provision of affordable spectacles will significantly reduce the national burden of vision loss in Australia.

[This corrects the article DOI: 10.1371/journal.pone.0152930.].

The Spumaretrovirinae, or foamy viruses (FVs) are complex retroviruses that infect many species of monkey and ape. Despite little sequence homology, FV and orthoretroviral Gag proteins perform equivalent functions, including genome packaging, virion assembly, trafficking and membrane targeting. However, there is a paucity of structural information for FVs and it is unclear how disparate FV and orthoretroviral Gag molecules share the same function. To probe the functional overlap of FV and orthoretroviral Gag we have determined the structure of a central region of Gag from the Prototype FV (PFV). The structure comprises two all α-helical domains NtDCEN and CtDCEN that although they have no sequence similarity, we show they share the same core fold as the N- (NtDCA) and C-terminal domains (CtDCA) of archetypal orthoretroviral capsid protein (CA). Moreover, structural comparisons with orthoretroviral CA align PFV NtDCEN and CtDCEN with NtDCA and CtDCA respectively. Further in vitro and functional virological assays reveal that residues making inter-domain NtDCEN—CtDCEN interactions are required for PFV capsid assembly and that intact capsid is required for PFV reverse transcription. These data provide the first information that relates the Gag proteins of Spuma and Orthoretrovirinae and suggests a common ancestor for both lineages containing an ancient CA fold., Author Summary Foamyviruses (FVs) or Spuma-retroviruses derive their name from the cytopathic effects they cause in cell culture. However, infection in humans is benign and FVs have entered the human population through zoonosis from apes resulting in the emergence of Prototype FV (PFV). Like all retroviruses, FVs contain gag, pol and env structural genes and replicate through reverse-transcription and host genome integration. Gag, the major structural protein, is required for genome packaging, virion assembly, trafficking and egress. However, although functionally equivalent, FV and orthoretroviral Gag share little sequence homology and it is unclear how they perform the same function. Therefore, to understand more about relationship between FV and orthoretroviral replication we have carried out structural studies of PFV-Gag. Here we present the structure of CA domains from a central region PFV-Gag and show that despite little sequence similarity they share the same fold as the CA domains of orthoretroviral Gag. These data provide the first information relating the Spuma and Orthoretrovirinae Gag proteins. We discuss our findings in terms of evolutionary divergence of spuma and orthoretroviral lineages.

Patients with biallelic truncating mutations in PALB2 have a severe form of Fanconi anaemia (FA-N), with a predisposition for developing embryonal-type tumours in infancy. Here we describe two unusual patients from a single family, carrying biallelic PALB2 mutations, one truncating, c.1676_1677delAAinsG;(p.Gln559ArgfsTer2), and the second, c.2586+1G>A; p.Thr839_Lys862del resulting in an in frame skip of exon 6 (24 amino acids). Strikingly, the affected individuals did not exhibit the severe developmental defects typical of FA-N patients and initially presented with B cell non-Hodgkin lymphoma. The expressed p.Thr839_Lys862del mutant PALB2 protein retained the ability to interact with BRCA2, previously unreported in FA-N patients. There was also a large increased chromosomal radiosensitivity following irradiation in G2 and increased sensitivity to mitomycin C. Although patient cells were unable to form Rad51 foci following exposure to either DNA damaging agent, U2OS cells, in which the mutant PALB2 with in frame skip of exon 6 was induced, did show recruitment of Rad51 to foci following damage. We conclude that a very mild form of FA-N exists arising from a hypomorphic PALB2 allele., Author Summary PALB2 is a protein that creates a molecular bridge that promotes the recruitment of Homologous Recombination Repair (HRR) proteins BRCA1, BRCA2 and Rad51 to sites of DNA damage. Cells with functional loss of PALB2 show a defect in HRR and are characterized by an increased number of spontaneous chromosome breaks and hypersensitivity to cross-linking agents. Typically, inherited mutations in PALB2 are associated with a severe Fanconi Anaemia phenotype. Here we describe for the first time the effect of biallelic mutations of PALB2 in which one hypomorphic allele resulted in a low level of expression of the mutant PALB2 protein with some retained function, as shown by its interaction with BRCA2, as well as its ability to facilitate Rad51 focus formation following damage. This resulted in a considerably milder clinical phenotype with increased longevity compared with biallelic PALB2 null patients and an altered tumour spectrum towards development of lymphoid malignancies.

Aim Cerebral palsy (CP) is associated with dysfunction of the upper motor neuron and results in balance problems and asymmetry during locomotion. Selective dorsal rhizotomy (SDR) is a surgical procedure that results in reduced afferent neuromotor signals from the lower extremities with the aim of improving gait. Its influence on balance and symmetry has not been assessed. The aim of this prospective cohort study was to evaluate the impact of SDR on balance and symmetry during walking. Methods 18 children (10 girls, 8 boys; age 6 years (y) 3 months (m), SD 1y 8m) with bilateral spastic CP and Gross Motor Function Classification System levels I to II underwent gait analysis before and 6 to 12 months after SDR. Results were compared to 11 typically developing children (TDC; 6 girls, 5 boys; age 6y 6m, SD 1y 11m). To analyse balance, sway velocity, radial displacement and frequency were calculated. Symmetry ratios were calculated for balance measures and spatio-temporal parameters during walking. Results Most spatio-temporal parameters of gait, as well as all parameters of balance, improved significantly after SDR. Preoperative values of symmetry did not vary considerably between CP and TDC group and significant postoperative improvement did not occur. Interpretation The reduction of afferent signalling through SDR improves gait by reducing balance problems rather than enhancing movement symmetry., PLoS ONE, 11 (4), ISSN:1932-6203

Chronic Hepatitis C Virus (HCV) infection is associated with progressive liver injury and subsequent development of fibrosis and cirrhosis. The death of hepatocytes results in the release of cytokines that induce inflammatory and fibrotic responses. The mechanism of liver damage is still under investigation but both apoptosis and immune-mediated processes may play roles. By observing the changes in gene expression patterns in HCV-infected cells, both markers and the causes of HCV-associated liver injury may be elucidated. HCV genotype 1b virus from persistently infected VeroE6 cells induced a strong cytopathic effect when used to infect Huh7.5 hepatoma cells. To determine if this cytopathic effect was a result of apoptosis, ultrastructural changes were observed by electron microscopy and markers of programmed cell death were surveyed. Screening of a human PCR array demonstrated a gene expression profile that contained upregulated markers of apoptosis, including tumor necrosis factor, caspases and caspase activators, Fas, Bcl2-interacting killer (BIK) and tumor suppressor protein, p53, as a result of HCV genotype 1b infection. The genes identified in this study should provide new insights into understanding viral pathogenesis in liver cells and may possibly help to identify novel antiviral and antifibrotic targets.

Many studies project future bird ranges by relying on correlative species distribution models. Such models do not usually represent important processes explicitly related to climate change and harvesting, which limits their potential for predicting and understanding the future of boreal bird assemblages at the landscape scale. In this study, we attempted to assess the cumulative and specific impacts of both harvesting and climate-induced changes on wildfires and stand-level processes (e.g., reproduction, growth) in the boreal forest of eastern Canada. The projected changes in these landscape- and stand-scale processes (referred to as "drivers of change") were then assessed for their impacts on future habitats and potential productivity of black-backed woodpecker (BBWO; Picoides arcticus), a focal species representative of deadwood and old-growth biodiversity in eastern Canada. Forest attributes were simulated using a forest landscape model, LANDIS-II, and were used to infer future landscape suitability to BBWO under three anthropogenic climate forcing scenarios (RCP 2.6, RCP 4.5 and RCP 8.5), compared to the historical baseline. We found climate change is likely to be detrimental for BBWO, with up to 92% decline in potential productivity under the worst-case climate forcing scenario (RCP 8.5). However, large declines were also projected under baseline climate, underlining the importance of harvest in determining future BBWO productivity. Present-day harvesting practices were the single most important cause of declining areas of old-growth coniferous forest, and hence appeared as the single most important driver of future BBWO productivity, regardless of the climate scenario. Climate-induced increases in fire activity would further promote young, deciduous stands at the expense of old-growth coniferous stands. This suggests that the biodiversity associated with deadwood and old-growth boreal forests may be greatly altered by the cumulative impacts of natural and anthropogenic disturbances under a changing climate. Management adaptations, including reduced harvesting levels and strategies to promote coniferous species content, may help mitigate these cumulative impacts.

Background Australia is the only high-income country in which endemic trachoma persists. In response, the Australian Government has recently invested heavily towards the nationwide control of the disease. Methodology/Principal Findings A novel simulation model was developed to reflect the trachoma epidemic in Australian Aboriginal communities. The model, which incorporates demographic, migration, mixing, and biological heterogeneities, was used to evaluate recent intervention measures against counterfactual past scenarios, and also to assess the potential impact of a series of hypothesized future intervention measures relative to the current national strategy and intensity. The model simulations indicate that, under the current intervention strategy and intensity, the likelihood of controlling trachoma to less than 5% prevalence among 5–9 year-old children in hyperendemic communities by 2020 is 31% (19%–43%). By shifting intervention priorities such that large increases in the facial cleanliness of children are observed, this likelihood of controlling trachoma in hyperendemic communities is increased to 64% (53%–76%). The most effective intervention strategy incorporated large-scale antibiotic distribution programs whilst attaining ambitious yet feasible screening, treatment, facial cleanliness and housing construction targets. Accordingly, the estimated likelihood of controlling trachoma in these communities is increased to 86% (76%–95%). Conclusions/Significance Maintaining the current intervention strategy and intensity is unlikely to be sufficient to control trachoma across Australia by 2020. However, by shifting the intervention strategy and increasing intensity, the likelihood of controlling trachoma nationwide can be significantly increased., Author Summary Australia is the only remaining high-income country reporting endemic levels of trachoma, with infections occurring predominantly within rural and remote Indigenous communities. Although the Australian government has recently invested large sums of money to combat the disease, it remains unclear whether the national goal of controlling trachoma by 2020 will be achieved. Here, we use a novel individual-based simulation model to estimate the impact of numerous potential future invention strategies and intensities. Our model is the most sophisticated trachoma transmission model to date, and the first to specifically represent trachoma in Australian Indigenous communities. Model projections suggest that although the current intervention strategy and intensity are unlikely to achieve the target of national control by 2020, the likelihood of achieving this goal can be significantly increased by shifting the intervention strategy and increasing the intensity of key intervention components such as screening, treatment and facial cleanliness activities. Our findings that the most resource rich country with endemic trachoma may require a more intensive intervention effort to control the disease suggest that challenges may remain in the fight for the global control and eventual elimination and eradication of trachoma.

The management of large animals during disasters and emergencies creates difficult operational environments for responders. The aims of this study were to identify the exact challenges faced by Australian emergency response personnel in their interactions with large animals and their owners, and to determine the readiness for large animal rescue (LAR) training in Australia. A survey tool collected the views and experiences of a broad cross section of emergency services personnel operating across Australia and across all hazards. Data were collected from 156 responders including Australian emergency services personnel, emergency managers such as federal agricultural departments, and local government. Overall, many of the respondents had serious concerns, and felt that there were significant issues in relation to LAR in Australia. These included the coordination of emergency care for animals, physical management of large animals, inter-agency coordination, and dealing with animal owners. Very few respondents had received any formal training in LAR, with an overwhelming majority indicating they would attend formal training if it were made available. Results help to guide the development of evidence-informed support tools to assist operational response and community engagement, and the production of professional development resources. Abstract The management of large animals during disasters and emergencies creates difficult operational environments for responders. The aims of this study were to identify the exact challenges faced by Australian emergency response personnel in their interactions with large animals and their owners, and to determine the readiness for large animal rescue (LAR) training in Australia. A survey tool collected the views and experiences of a broad cross section of emergency services personnel operating across Australia and across all hazards. Data were collected from 156 responders including Australian emergency services personnel, emergency managers such as federal agricultural departments, and local government. Overall, many of the respondents had serious concerns, and felt that there were significant issues in relation to LAR in Australia. These included the coordination of emergency care for animals, physical management of large animals, inter-agency coordination, and dealing with animal owners. Very few respondents had received any formal training in LAR, with an overwhelming majority indicating they would attend formal training if it were made available. Results help to guide the development of evidence-informed support tools to assist operational response and community engagement, and the production of professional development resources. Abstract The management of large animals during disasters and emergencies creates difficult operational environments for responders. The aims of this study were to identify the exact challenges faced by Australian emergency response personnel in their interactions with large animals and their owners, and to determine the readiness for large animal rescue (LAR) training in Australia. A survey tool collected the views and experiences of a broad cross section of emergency services personnel operating across Australia and across all hazards. Data were collected from 156 responders including Australian emergency services personnel, emergency managers such as federal agricultural departments, and local government. Overall, many of the respondents had serious concerns, and felt that there were significant issues in relation to LAR in Australia. These included the coordination of emergency care for animals, physical management of large animals, inter-agency coordination, and dealing with animal owners. Very few respondents had received any formal training in LAR, with an overwhelming majority indicating they would attend formal training if it were made available. Results help to guide the development of evidence-informed support tools to assist operational response and community engagement, and the production of professional development resources.

With the rapidly increasing abundance and accessibility of genomic data, there is a growing interest in using population genetic approaches to characterize fine-scale dispersal of organisms, providing insight into biological processes across a broad range of fields including ecology, evolution and epidemiology. For sexually recombining haploid organisms such as the human malaria parasite P. falciparum, however, there have been no systematic assessments of the type of data and methods required to resolve fine scale connectivity. This analytical gap hinders the use of genomics for understanding local transmission patterns, a crucial goal for policy makers charged with eliminating this important human pathogen. Here we use data collected from four clinics with a catchment area spanning approximately 120 km of the Thai-Myanmar border to compare the ability of divergence (FST) and relatedness based on identity by descent (IBD) to resolve spatial connectivity between malaria parasites collected from proximal clinics. We found no relationship between inter-clinic distance and FST, likely due to sampling of highly related parasites within clinics, but a significant decline in IBD-based relatedness with increasing inter-clinic distance. This association was contingent upon the data set type and size. We estimated that approximately 147 single-infection whole genome sequenced parasite samples or 222 single-infection parasite samples genotyped at 93 single nucleotide polymorphisms (SNPs) were sufficient to recover a robust spatial trend estimate at this scale. In summary, surveillance efforts cannot rely on classical measures of genetic divergence to measure P. falciparum transmission on a local scale. Given adequate sampling, IBD-based relatedness provides a useful alternative, and robust trends can be obtained from parasite samples genotyped at approximately 100 SNPs., Author summary The spatiotemporal dispersal of organisms can inform efforts to conserve endangered species, to contain the spread of drug resistance, and to eliminate disease. As genomic data become increasingly more affordable and accessible via public depositories, the demand for methods capable of extracting fine-scale population structure from genomic data grows. However, to the best of our knowledge, there are no guidelines regarding the type of data and methods required to resolve local spatial trends over sexually recombining haploid organisms, such as the malaria parasite. The approach we present here compares relatedness based on identity by descent, which accounts for recombination while distinguishing genetic identity due to inheritance from genetic identity due to chance, to a classic population genetic measure of divergence, using data from sexually recombining malaria parasites. Using identity by descent, we uncover a significant decrease in highly related malaria parasites collected from proximal clinics on the Thai-Myanmar border, a region where human mobility is high. Our results demonstrate the power of analyses based on identity by descent to detect recent and local trends. Similar analyses could be used to inform the molecular epidemiology of other sexually recombining organisms.

To assess the change in the size of the eastern North American monarch butterfly summer population, studies have used long-term data sets of counts of adult butterflies or eggs per milkweed stem. Despite the observed decline in the monarch population as measured at overwintering sites in Mexico, these studies found no decline in summer counts in the Midwest, the core of the summer breeding range, leading to a suggestion that the cause of the monarch population decline is not the loss of Midwest agricultural milkweeds but increased mortality during the fall migration. Using these counts to estimate population size, however, does not account for the shift of monarch activity from agricultural fields to non-agricultural sites over the past 20 years, as a result of the loss of agricultural milkweeds due to the near-ubiquitous use of glyphosate herbicides. We present the counter-hypotheses that the proportion of the monarch population present in non-agricultural habitats, where counts are made, has increased and that counts reflect both population size and the proportion of the population observed. We use data on the historical change in the proportion of milkweeds, and thus monarch activity, in agricultural fields and non-agricultural habitats to show why using counts can produce misleading conclusions about population size. We then separate out the shifting proportion effect from the counts to estimate the population size and show that these corrected summer monarch counts show a decline over time and are correlated with the size of the overwintering population. In addition, we present evidence against the hypothesis of increased mortality during migration. The milkweed limitation hypothesis for monarch decline remains supported and conservation efforts focusing on adding milkweeds to the landscape in the summer breeding region have a sound scientific basis.

Purpose To present the rates of referral of participants in the National Eye Health Survey (NEHS) for further eye care. Materials & methods A national sample of 3098 non-Indigenous Australians aged 50–98 and 1738 Indigenous Australians aged 40–92 years living in 30 randomly selected sites was recruited using a door-to-door approach. Participants completed a general questionnaire and a series of eye tests, including vision and anterior segment assessment, intra-ocular pressure measurement, visual field testing and fundus photography. A predefined protocol was used to guide the referral of participants for follow up eye care. An ophthalmologist was on-call to assist with the triaging of participants. Results Of the total sample, 32.1% (994/3098) of non-Indigenous participants and 43.6% (757/1738) of Indigenous participants were referred for further eye care (p

BACKGROUND:Monoclonal antibodies (mAbs) which potently neutralize a broad range of HIV isolates are potential microbicide candidates. To date, topical application of mAbs in humans and their stability in vaginal secretions has not been studied. OBJECTIVES:To assess the pharmacokinetics and safety of the mAbs 2F5, 4E10 and 2G12 when applied vaginally in women. DESIGN:A randomized, double-blind, placebo-controlled phase 1 trial. METHODS:Twenty-eight healthy, sexually abstinent women administered 2.5 g of gel daily for 12 days containing either 10 or 20 mg/g of each mAb (MABGEL) or placebo. Main clinical evaluations and sampling occurred at baseline, 1, 8, and 24 hours post-1st dose and 12 and 36 hours post-12th dose. RESULTS:After adjustment for dilution factors, median levels of 2F5, 4E10 and 2G12 in vaginal secretions at 1 hour post high-dose MABGEL were 7.74, 5.28 and 7.48 mg/ml respectively. Levels of 2F5 and 4E10 declined exponentially thereafter with similar estimated half-lives (4.6 and 4.3 hours). In contrast, 2G12 levels declined more rapidly in the first 8 hours, with an estimated half-life of 1.4 hours during this period. There was no evidence of systemic absorption. There were no significant differences in local or systemic adverse event rates or vaginal flora changes (by qPCR) between active and placebo gel arms. Whilst at least 1 adverse event was recorded in 96% of participants, 95% were mild and none were serious. CONCLUSIONS:Vaginal application of 50 mg of each mAb daily was safe over a 12 day period. Median mAb concentrations detected at 8 hours post dose were potentially sufficient to block HIV transmission.2G12 exhibited more rapid elimination from the human vagina than 4E10 and 2F5, likely due to poor stability of 2G12 in acidic human vaginal secretions. Further research is needed to develop mAb-based vaginal microbicides and delivery systems. TRIAL REGISTRATION:ISRCTN 64808733 UK CRN Portfolio 6470.

Population genetic differentiation will be influenced by the demographic history of populations, opportunities for migration among neighboring demes and founder effects associated with repeated extinction and recolonization. In natural populations, these factors are expected to interact with each other and their magnitudes will vary depending on the spatial distribution and age structure of local demes. Although each of these effects has been individually identified as important in structuring genetic variance, their relative magnitude is seldom estimated in nature. We conducted a population genetic analysis in a metapopulation of the angiosperm, Silene latifolia, from which we had more than 20 years of data on the spatial distribution, demographic history, and extinction and colonization of demes. We used hierarchical Bayesian methods to disentangle which features of the populations contributed to among population variation in allele frequencies, including the magnitude and direction of their effects. We show that population age, long-term size and degree of connectivity all combine to affect the distribution of genetic variance; small, recently-founded, isolated populations contributed most to increase F ST in the metapopulation. However, the effects of population size and population age are best understood as being modulated through the effects of connectivity to other extant populations, i.e. F ST diminishes as populations age, but at a rate that depends how isolated the population is. These spatial and temporal correlates of population structure give insight into how migration, founder effect and within-deme genetic drift have combined to enhance and restrict genetic divergence in a natural metapopulation.

Purpose: Retinal dystrophies are genetically heterogeneous, resulting from mutations in over 200 genes. Prior to the development of massively parallel sequencing, comprehensive genetic screening was unobtainable for most patients. Identifying the causative genetic mutation facilitates genetic counselling, carrier testing and prenatal/pre-implantation diagnosis, and often leads to a clearer prognosis. In addition, in a proportion of cases, when the mutation is known treatment can be optimised and patients are eligible for enrolment into clinical trials for gene-specific therapies. Methods: Patient genomic DNA was sheared, tagged and pooled in batches of four samples, prior to targeted capture and next generation sequencing. The enrichment reagent was designed against genes listed on the RetNet database (July 2010). Sequence data were aligned to the human genome and variants were filtered to identify potential pathogenic mutations. These were confirmed by Sanger sequencing. Results: Molecular analysis of 20 DNAs from retinal dystrophy patients identified likely pathogenic mutations in 12 cases, many of them known and/or confirmed by segregation. These included previously described mutations in ABCA4 (c.6088C> T,p.R2030*; c.5882G>A,p.G1961E), BBS2 (c.1895G>C,p.R632P), GUCY2D (c.2512C>T,p.R838C), PROM1 (c.1117C>T,p.R373C), RDH12 (c.601T>C,p. C201R; c.506G>A,p.R169Q), RPGRIP1 (c.3565C>T,p.R1189*) and SPATA7 (c.253C>T,p.R85*) and new mutations in ABCA4 (c.3328+1G>C), CRB1 (c.2832-2842+23del), RP2 (c.884-1G>T) and USH2A (c.12874A>G,p.N4292D). Conclusions: Tagging and pooling DNA prior to targeted capture of known retinal dystrophy genes identified mutations in 60% of cases. This relatively high success rate may reflect enrichment for consanguineous cases in the local Yorkshire population, and the use of multiplex families. Nevertheless this is a promising high throughput approach to retinal dystrophy diagnostics.

Background: Barrett’s esophagus (BE) is a metaplastic precursor lesion of esophageal adenocarcinoma (EA), the most rapidly increasing cancer in western societies. While the prevalence of BE is increasing, the vast majority of EA occurs in patients with undiagnosed BE. Thus, we sought to identify genes that are altered in BE compared to the normal mucosa of the esophagus, and which may be potential biomarkers for the development or diagnosis of BE. Design: We performed gene expression analysis using HG-U133A Affymetrix chips on fresh frozen tissue samples of Barrett’s metaplasia and matched normal mucosa from squamous esophagus (NE) and gastric cardia (NC) in 40 BE patients. Results: Using a cut off of 2-fold and P,1.12E-06 (0.05 with Bonferroni correction), we identified 1324 differentiallyexpressed genes comparing BE vs NE and 649 differentially-expressed genes comparing BE vs NC. Except for individual genes such as the SOXs and PROM1 that were dysregulated only in BE vs NE, we found a subset of genes (n=205) whose expression was significantly altered in both BE vs NE and BE vs NC. These genes were overrepresented in different pathways, including TGF-b and Notch. Conclusion: Our findings provide additional data on the global transcriptome in BE tissues compared to matched NE and NC tissues which should promote further understanding of the functions and regulatory mechanisms of genes involved in BE development, as well as insight into novel genes that may be useful as potential biomarkers for the diagnosis of BE in the future.

The intracellular mechanisms underlying memory reconsolidation critically involve cAMP signaling. These events were originally attributed to PKA activation by cAMP, but the identification of Exchange Protein Activated by cAMP (Epac), as a distinct mediator of cAMP signaling, suggests that cAMP-regulated processes that subserve memory reconsolidation are more complex. Here we investigated how activation of Epac with 8-pCPT-cAMP (8-CPT) impacts reconsolidation of a memory that had been associated with cocaine self-administration. Rats were trained to lever press for cocaine on an FR-1 schedule, in which each cocaine delivery was paired with a tone+light cue. Lever pressing was then extinguished in the absence of cue presentations and cocaine delivery. Following the last day of extinction, rats were put in a novel context, in which the conditioned cue was presented to reactivate the cocaine-associated memory. Immediate bilateral infusions of 8-CPT into the basolateral amygdala (BLA) following reactivation disrupted subsequent cue-induced reinstatement in a dose-dependent manner, and modestly reduced responding for conditioned reinforcement. When 8-CPT infusions were delayed for 3 hours after the cue reactivation session or were given after a cue extinction session, no effect on cue-induced reinstatement was observed. Co-administration of 8-CPT and the PKA activator 6-Bnz-cAMP (10 nmol/side) rescued memory reconsolidation while 6-Bnz alone had no effect, suggesting an antagonizing interaction between the two cAMP signaling substrates. Taken together, these studies suggest that activation of Epac represents a parallel cAMP-dependent pathway that can inhibit reconsolidation of cocaine-cue memories and reduce the ability of the cue to produce reinstatement of cocaine-seeking behavior.

Objectives Precursors to 1/3 of colorectal cancer (CRC), serrated polyps have been under-detected by screening due to their inconspicuous, non-hemorrhagic, and proximal nature. A new multi-target stool DNA test (multi-target sDNA) shows high sensitivity for both CRC and advanced adenomas. Screen detection of serrated polyps by this approach requires further validation. We sought to assess and compare noninvasive detection of sessile serrated polyps (SSP) ≥1 cm by sDNA and an occult blood fecal immunochemical test (FIT). Methods In a blinded prospective study, a single stool sample used for both tests was collected from 456 asymptomatic adults prior to screening or surveillance colonoscopy (criterion standard). All 29 patients with SSP≥1 cm were included as cases and all 232 with no neoplastic findings as controls. Buffered stool samples were processed and frozen on receipt; Exact Sciences performed sDNA in batches using optimized analytical methods. The sDNA multi-marker panel targets methylated BMP3 (mBMP3) and NDRG4, mutant KRAS, β-actin, and hemoglobin. FIT (Polymedco OC-FIT Check) was performed in separate lab ≤2 days post defecation and evaluated at cutoffs of 50 (FIT-50) and 100 ng/ml (FIT-100). Results Median ages: cases 61 (range 57–77), controls 62 (52–70), p = NS. Women comprised 59% and 51%, p = NS, respectively. SSP median size was 1.2 cm (1–3 cm), 93% were proximal, and 64% had synchronous diminutive polyps. Among multi-target sDNA markers, mBMP3 proved highly discriminant for detection of SSP≥1 cm (AUC = 0.87, p

Chronic exposure to stress has been widely implicated in the development of anxiety disorders, yet relatively little is known about the long-term effects of chronic stress on amygdala-dependent memory formation. Here, we examined the effects of a history of chronic exposure to the stress-associated adrenal steroid corticosterone (CORT) on the consolidation of a fear memory and the expression of memory-related immediate early genes (IEGs) in the lateral nucleus of the amygdala (LA). Rats received chronic exposure to CORT (50 μg/ml) in their drinking water for 2 weeks and were then titrated off the CORT for an additional 6 days followed by a 2 week 'wash-out' period consisting of access to plain water. Rats were then either sacrificed to examine the expression of memory-related IEG expression in the LA or given auditory Pavlovian fear conditioning. We show that chronic exposure to CORT leads to a persistent elevation in the expression of the IEGs Arc/Arg3.1 and Egr-1 in the LA. Further, we show that rats with a history of chronic CORT exposure exhibit enhanced consolidation of a fear memory; short-term memory (STM) is not affected, while long-term memory (LTM) is significantly enhanced. Treatment with the selective serotonin reuptake inhibitor (SSRI) fluoxetine following the chronic CORT exposure period was observed to effectively reverse both the persistent CORT-related increases in memory-related IEG expression in the LA and the CORT-related enhancement in fear memory consolidation. Our findings suggest that chronic exposure to CORT can regulate memory-related IEG expression and fear memory consolidation processes in the LA in a long-lasting manner and that treatment with fluoxetine can reverse these effects.

Objective To profile RNA expression in gastric cancer by anatomic subsites as an initial step in identifying molecular subtypes and providing targets for early detection and therapy. Methods We performed transcriptome analysis using the Affymetrix GeneChip U133A in gastric cardia adenocarcinomas (n = 62) and gastric noncardia adenocarcinomas (n = 72) and their matched normal tissues from patients in Shanxi Province, and validated selected dysregulated genes with additional RNA studies. Expression of dysregulated genes was also related to survival of cases. Results Principal Component Analysis showed that samples clustered by tumor vs. normal, anatomic location, and histopathologic features. Paired t-tests of tumor/normal tissues identified 511 genes whose expression was dysregulated (P

Bone Morphogenetic Proteins (BMPs) are morphogens that play a major role in regulating development and homeostasis. Although BMPs are used for the treatment of bone and kidney disorders, their clinical use is limited due to the supra-physiological doses required for therapeutic efficacy causing severe side effects. Because recombinant BMPs are expensive to produce, small molecule activators of BMP signaling would be a cost-effective alternative with the added benefit of being potentially more easily deliverable. Here, we report our efforts to identify small molecule activators of BMP signaling. We have developed a cell-based assay to monitor BMP signaling by stably transfecting a BMP-responsive human cervical carcinoma cell line (C33A) with a reporter construct in which the expression of luciferase is driven by a multimerized BMP-responsive element from the Id1 promoter. A BMP-responsive clone C33A-2D2 was used to screen a bioactive library containing ∼5,600 small molecules. We identified four small molecules of the family of flavonoids all of which induced luciferase activity in a dose-dependent manner and ventralized zebrafish embryos. Two of the identified compounds induced Smad1, 5 phosphorylation (P-Smad), Id1 and Id2 expression in a dose-dependent manner demonstrating that our assays identified small molecule activators of BMP signaling.

BACKGROUND: Chronic glucocorticoid excess has been linked to increased atherosclerosis and general cardiovascular risk in humans. The enzyme 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1) increases active glucocorticoid levels within tissues by catalyzing the conversion of cortisone to cortisol. Pharmacological inhibition of 11βHSD1 has been shown to reduce atherosclerosis in murine models. However, the cellular and molecular details for this effect have not been elucidated. METHODOLOGY/PRINCIPAL FINDINGS: To examine the role of 11βHSD1 in atherogenesis, 11βHSD1 knockout mice were created on the pro-atherogenic apoE⁻/⁻ background. Following 14 weeks of Western diet, aortic cholesterol levels were reduced 50% in 11βHSD1⁻/⁻/apoE⁻/⁻ mice vs. 11βHSD1⁺/⁺/apoE⁻/⁻ mice without changes in plasma cholesterol. Aortic 7-ketocholesterol content was reduced 40% in 11βHSD1⁻/⁻/apoE⁻/⁻ mice vs. control. In the aortic root, plaque size, necrotic core area and macrophage content were reduced ∼30% in 11βHSD1⁻/⁻/apoE⁻/⁻mice. Bone marrow transplantation from 11βHSD1⁻/⁻/apoE⁻/⁻ mice into apoE⁻/⁻ recipients reduced plaque area 39-46% in the thoracic aorta. In vivo foam cell formation was evaluated in thioglycollate-elicited peritoneal macrophages from 11βHSD1⁺/⁺/apoE⁻/⁻ and 11βHSD1⁻/⁻/apoE⁻/⁻ mice fed a Western diet for ∼5 weeks. Foam cell cholesterol levels were reduced 48% in 11βHSD1⁻/⁻/apoE⁻/⁻ mice vs. control. Microarray profiling of peritoneal macrophages revealed differential expression of genes involved in inflammation, stress response and energy metabolism. Several toll-like receptors (TLRs) were downregulated in 11βHSD1⁻/⁻/apoE⁻/⁻ mice including TLR 1, 3 and 4. Cytokine release from 11βHSD1⁻/⁻/apoE⁻/⁻-derived peritoneal foam cells was attenuated following challenge with oxidized LDL. CONCLUSIONS: These findings suggest that 11βHSD1 inhibition may have the potential to limit plaque development at the vessel wall and regulate foam cell formation independent of changes in plasma lipids. The diminished cytokine response to oxidized LDL stimulation is consistent with the reduction in TLR expression and suggests involvement of 11βHSD1 in modulating binding of pro-atherogenic TLR ligands.

We show that by using a combination of computational methods, consistent three-dimensional molecular models can be proposed for the core proteins of the type-III secretion system. We employed a variety of approaches to reconcile disparate, and sometimes inconsistent, data sources into a coherent picture that for most of the proteins indicated a unique solution to the constraints. The range of difficulty spanned from the trivial (FliQ) to the difficult (FlhA and FliP). The uncertainties encountered with FlhA were largely the result of the greater number of helix packing possibilities allowed in a large protein, however, for FliP, there remains an uncertainty in how to reconcile the large displacement predicted between its two main helical hairpins and their ability to sit together happily across the bacterial membrane. As there is still no high resolution structural information on any of these proteins, we hope our predicted models may be of some use in aiding the interpretation of electron microscope images and in rationalising mutation data and experiments.

OTSSP167 was recently characterized as a potent inhibitor for maternal embryonic leucine zipper kinase (MELK) and is currently tested in Phase I clinical trials for solid tumors that have not responded to other treatment. Here we report that OTSSP167 abrogates the mitotic checkpoint at concentrations used to inhibit MELK. The abrogation is not recapitulated by RNAi mediated silencing of MELK in cells. Although OTSSP167 indeed inhibits MELK, it exhibits off-target activity against Aurora B kinase in vitro and in cells. Furthermore, OTSSP167 inhibits BUB1 and Haspin kinases, reducing phosphorylation at histones H2AT120 and H3T3 and causing mislocalization of Aurora B and associated chromosomal passenger complex from the centromere/kinetochore. The results suggest that OTSSP167 may have additional mechanisms of action for cancer cell killing and caution the use of OTSSP167 as a MELK specific kinase inhibitor in biochemical and cellular assays.

Poor outcome in response to hepatitis C virus, including higher viral load, hepatocellular carcinoma and cirrhosis, is more associated with men and postmenopausal women than with premenopausal women and women receiving hormone replacement therapy, suggesting that β-estradiol plays an innate role in preventing viral infection and liver disease. Consequently, most research in the field has concluded that estrogen affects HCV replication through viral interactions with estrogen receptor-α. Previously, estrogen-like antagonists, including Tamoxifen, were shown to reduce HCV RNA production and prevent viral entry, although the authors did not identify host factors involved. Estrogen can act alternatively through the membrane-bound G-protein-coupled estrogen receptor, GPR30. Here, human hepatoma Huh7.5 cells were infected with HCV J6/JFH-1 and treated with estrogen or Tamoxifen, resulting in a marked decrease in detectable virus. The effect was mimicked by G1, a GPR30-specific agonist, and was reversed by the GPR30-specific antagonist, G15. While previous studies have demonstrated that estrogen down-regulated occludin in cervical cancer cells, its action on liver cells was unknown. Occludin is a tight junction protein and HCV receptor and here we report that activation and cellular export of MMP-9 led to the cleavage of occludin upon estrogen treatment of liver cells. This is the first report of the cleavage of an HCV receptor in response to estrogen. We also identify the occludin cleavage site in extracellular Domain D; the motif required for HCV entry and spread. This pathway gives new insight into a novel innate antiviral pathway and the suboptimal environment that estrogen provides for the proliferation of the virus. It may also explain the disparate host-virus responses to HCV demonstrated by the two sexes. Moreover, these data suggest that hormone replacement therapy may have beneficial antiviral enhancement properties for HCV-infected postmenopausal women and show promise for new antiviral treatments for both men and women.

A general understanding of the complex phenomenon of protein evolution requires the accurate description of the constraints that define the sub-space of proteins with mutations that do not appreciably reduce the fitness of the organism. Such constraints can have multiple origins, in this work we present a model for constrained evolutionary trajectories represented by a markovian process throughout a set of protein-like structures artificially constructed to be topological intermediates between the structure of two natural occurring proteins. The number and type of intermediate steps defines how constrained the total evolutionary process is. By using a coarse-grained representation for the protein structures, we derive an analytic formulation of the transition rates between each of the intermediate structures. The results indicate that compact structures with a high number of hydrogen bonds are more probable and have a higher likelihood to arise during evolution. Knowledge of the transition rates allows for the study of complex evolutionary pathways represented by trajectories through a set of intermediate structures.

From simulations that begin with a random mix of two cell types, we monitor progress towards segregation driven by contact-mediated linkage of model cells, which is equivalent to the cell-cell adhesion of real cells. In comparison with real cell experiments, we show that this mechanical model can account for the observed extent of segregation obtained by differential adhesion in a 2D cell culture assay of cells with differentially expressed cadherin molecules. Calibration of virtual to real time allowed us to estimate a time course for these experiments that was within 50% agreement for the simulations compared to differential adhesion of cells. In contrast, simulations of differential adhesion do not account for the rate of segregation driven by interactions between EphB2 receptor and ephrinB1 expressing cells which occurs an order of magnitude faster. The latter result suggests that mechanisms additional or alternative to differential adhesion contribute to Eph-ephrin mediated cell segregation.

Photosystem I is the light-driven plastocyanin-ferredoxin oxidoreductase in the photosynthetic electron transfer of cyanobacteria and plants. Two histidyl residues in the symmetric transmembrane helices A-j and B-j provide ligands for the P700 chlorophyll molecules of the reaction center of photosystem I. To determine the role of conserved aromatic residues adjacent to the histidyl molecule in the helix of B-j, we generated six site-directed mutants of the psaB gene in Synechocystis sp. PCC 6803. Three mutant strains with W645C, W643C/A644I and S641C/V642I substitutions could grow photoautotrophically and showed no obvious reduction in the photosystem I activity. Kinetics of P700 re-reduction by plastocyanin remained unaltered in these mutants. In contrast, the strains with H651C/L652M, F649C/G650I and F647C substitutions could not grow under photoautotrophic conditions because those mutants had low photosystem I activity, possibly due to low levels of proteins. A procedure to select spontaneous revertants from the mutants that are incapable to photoautotrophic growth resulted in three revertants that were used in this study. The molecular analysis of the spontaneous revertants suggested that an aromatic residue at F647 and a small residue at G650 may be necessary for maintaining the structural integrity of photosystem I. The (P700+ - P700) steady-state absorption difference spectrum of the revertant F647Y has a ∼5 nm narrower peak than the recovered wild-type, suggesting that additional hydroxyl group of this revertant may participate in the interaction with the special pair while the photosystem I complexes of the F649C/G650T and H651Q mutants closely resemble the wild-type spectrum. The results presented here demonstrate that the highly conserved residues W645, W643 and F649 are not critical for maintaining the integrity and in mediating electron transport from plastocyanin to photosystem I. Our data suggest that an aromatic residue is required at position of 647 for structural integrity and/or function of photosystem I.

In prion diseases, the cellular form of the prion protein, PrPC, undergoes a conformational conversion to the infectious isoform, PrPSc. PrPC associates with lipid rafts through its glycosyl-phosphatidylinositol (GPI) anchor and a region in its N-terminal domain which also binds to heparan sulfate proteoglycans (HSPGs). We show that heparin displaces PrPC from rafts and promotes its endocytosis, suggesting that heparin competes with an endogenous raft-resident HSPG for binding to PrPC. We then utilised a transmembrane-anchored form of PrP (PrP-TM), which is targeted to rafts solely by its N-terminal domain, to show that both heparin and phosphatidylinositol-specific phospholipase C can inhibit its association with detergent-resistant rafts, implying that a GPI-anchored HSPG targets PrPC to rafts. Depletion of the major neuronal GPI-anchored HSPG, glypican-1, significantly reduced the raft association of PrP-TM and displaced PrPC from rafts, promoting its endocytosis. Glypican-1 and PrPC colocalised on the cell surface and both PrPC and PrPSc co-immunoprecipitated with glypican-1. Critically, treatment of scrapie-infected N2a cells with glypican-1 siRNA significantly reduced PrPSc formation. In contrast, depletion of glypican-1 did not alter the inhibitory effect of PrPC on the β-secretase cleavage of the Alzheimer's amyloid precursor protein. These data indicate that glypican-1 is a novel cellular cofactor for prion conversion and we propose that it acts as a scaffold facilitating the interaction of PrPC and PrPSc in lipid rafts., Author Summary The prion diseases are unique in that their infectious nature is not dependent on nucleic acid but is instead attributed to a misfolded protein, the prion protein. This misfolded prion protein is capable of inducing the misfolding of the normal form of the prion protein that is present on the surface of neurons and other cells in the body. However, the site in the cell at which this misfolding occurs and whether other proteins are involved remains controversial. We have addressed these questions by investigating how the normal form of the prion protein is targeted to specialised domains on the plasma membrane termed cholesterol-rich lipid rafts. We show that targeting is due, in part, to a particular heparin sulfate proteoglycan called glypican-1. Significantly, reducing the levels of glypican-1 in an infected cell line reduced the accumulation of misfolded prion protein. We propose that glypican-1 acts as a scaffold facilitating the favourable interaction of the misfolded, infectious form of the prion protein with the normal cellular form within cholesterol-rich lipid rafts. Our results indicate that glypican-1 is intimately involved in the misfolding of the prion protein, the critical event in the pathogenesis of prion diseases such as Creutzfeldt-Jakob disease in humans.

Professional soccer players have a lengthy playing season, throughout which high levels of physical stress are maintained. The following recuperation period, before starting the next pre-season training phase, is generally considered short but sufficient to allow a decrease in these stress levels and therefore a reduction in the propensity for injury or musculoskeletal tissue damage. We hypothesised that these physical extremes influence the body composition, blood flow, and endothelial/immune function, but that the recuperation may be insufficient to allow a reduction of tissue stress damage. Ten professional football players were examined at the end of the playing season, at the end of the season intermission, and after the next pre-season endurance training. Peripheral blood flow and body composition were assessed using venous occlusion plethysmography and DEXA scanning respectively. In addition, selected inflammatory and immune parameters were analysed from blood samples. Following the recuperation period a significant decrease of lean body mass from 74.4+/-4.2 kg to 72.2+/-3.9 kg was observed, but an increase of fat mass from 10.3+/-5.6 kg to 11.1+/-5.4 kg, almost completely reversed the changes seen in the pre-season training phase. Remarkably, both resting and post-ischemic blood flow (7.3+/-3.4 and 26.0+/-6.3 ml/100 ml/min) respectively, were strongly reduced during the playing and training stress phases, but both parameters increased to normal levels (9.0+/-2.7 and 33.9+/-7.6 ml/100 ml/min) during the season intermission. Recovery was also characterized by rising levels of serum creatinine, granulocytes count, total IL-8, serum nitrate, ferritin, and bilirubin. These data suggest a compensated hypo-perfusion of muscle during the playing season, followed by an intramuscular ischemia/reperfusion syndrome during the recovery phase that is associated with muscle protein turnover and inflammatory endothelial reaction, as demonstrated by iNOS and HO-1 activation, as well as IL-8 release. The data provided from this study suggest that the immune system is not able to function fully during periods of high physical stress. The implications of this study are that recuperation should be carefully monitored in athletes who undergo intensive training over extended periods, but that these parameters may also prove useful for determining an individual's risk of tissue stress and possibly their susceptibility to progressive tissue damage or injury.

Activation-induced cytidine deaminase (AID) is believed to initiate somatic hypermutation (SHM) by deamination of deoxycytidines to deoxyuridines within the immunoglobulin variable regions genes. The deaminated bases can subsequently be replicated over, processed by base excision repair or mismatch repair, leading to introduction of different types of point mutations (G/C transitions, G/C transversions and A/T mutations). It is evident that the base excision repair pathway is largely dependent on uracil-DNA glycosylase (UNG) through its uracil excision activity. It is not known, however, which endonuclease acts in the step immediately downstream of UNG, i.e. that cleaves at the abasic sites generated by the latter. Two candidates have been proposed, an apurinic/apyrimidinic endonuclease (APE) and the Mre11-Rad50-NBS1 complex. The latter is intriguing as this might explain how the mutagenic pathway is primed during SHM. We have investigated the latter possibility by studying the in vivo SHM pattern in B cells from ataxia-telangiectasia-like disorder (Mre11 deficient) and Nijmegen breakage syndrome (NBS1 deficient) patients. Our results show that, although the pattern of mutations in the variable heavy chain (V(H)) genes was altered in NBS1 deficient patients, with a significantly increased number of G (but not C) transversions occurring in the SHM and/or AID targeting hotspots, the general pattern of mutations in the V(H) genes in Mre11 deficient patients was only slightly altered, with an increased frequency of A to C transversions. The Mre11-Rad50-NBS1 complex is thus unlikely to be the major nuclease involved in cleavage of the abasic sites during SHM, whereas NBS1 might have a specific role in regulating the strand-biased repair during phase Ib mutagenesis.

Several recent studies have shown a genetic influence on gene expression variation, including variation between the two chromosomes within an individual and variation between individuals at the population level. We hypothesized that genetic inheritance may also affect variation in chromatin states. To test this hypothesis, we analyzed chromatin states in 12 lymphoblastoid cells derived from two Centre d'Etude du Polymorphisme Humain families using an allele-specific chromatin immunoprecipitation (ChIP-on-chip) assay with Affymetrix 10K SNP chip. We performed the allele-specific ChIP-on-chip assays for the 12 lymphoblastoid cells using antibodies targeting at RNA polymerase II and five post-translation modified forms of the histone H3 protein. The use of multiple cell lines from the Centre d'Etude du Polymorphisme Humain families allowed us to evaluate variation of chromatin states across pedigrees. These studies demonstrated that chromatin state clustered by family. Our results support the idea that genetic inheritance can determine the epigenetic state of the chromatin as shown previously in model organisms. To our knowledge, this is the first demonstration in humans that genetics may be an important factor that influences global chromatin state mediated by histone modification, the hallmark of the epigenetic phenomena., Author Summary Human health and disease are determined by an interaction between genetic background and environmental exposures. Both normal development and disease are mediated by epigenetic regulation of gene expression. The epigenetic regulation causes heritable changes in gene expression, which is not associated with DNA sequence changes. Instead, it is mediated by chemical modification of DNA such as DNA methylation or by protein modifications such as histone acetylation and methylation. Although much has been known about epigenetic inheritance during development, little is known about the influence of the genetic background on epigenetic processes such as histone modifications. In this report the authors studied five histone modifications on a genome-wide level in cells from different families. Global epigenetic states, as measured by these histone modifications, showed a similar pattern for cells derived from the same family. This study demonstrates that genetic inheritance may be an important factor influencing global chromatin states mediated by histone modifications in humans. These observations illustrate the importance of integrating genetic and epigenetic information into studies of human health and complex diseases.